Synthetic HIV gag genes

ABSTRACT

Synthetic DNA molecules encoding HIV gag and modifications of HIV gag are provided. The codons of the synthetic molecules are codons preferred by the projected host cell. The synthetic molecules may be used as a polynucleotide vaccine which provides effective immunoprophylaxis against HIV infection through stimulation of neutralizing antibody and cell-mediated immunity.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of co-pending U.S. applicationSer. No. 09/974,702, filed Oct. 9, 2001, which is a continuation ofco-pending U.S. application Ser. No. 09/340,871, filed Jun. 28, 1999,which is a continuation of co-pending U.S. application Ser. No.09/017,981, filed Feb. 3, 1998, now abandoned, which claims benefit ofU.S. Provisional application serial no. 60/037,854, filed Feb. 7, 1997.The benefits of priority under 35 USC §§ 119 and 120, as applicable, areclaimed for all the foregoing applications, and each such application ishereby incorporated by reference into the present application.

FIELD OF INVENTION

[0002] HIV vaccines.

BACKGROUND OF THE INVENTION

[0003] Human Immunodeficiency Virus- 1 (HIV-1) is the etiological agentof acquired human immune deficiency syndrome (AIDS) and relateddisorders. HIV-1 is an RNA virus of the Retroviridae family and exhibitsthe 5′LTR-gag-pol-env-LTR3′ organization of all retroviruses. Inaddition, HIV- 1 comprises a handful of genes with regulatory or unknownfunctions, including the tat and rev genes. The env gene encodes theviral envelope glycoprotein that is translated as a 160-kilodalton (kDa)precursor (gp 160) and then cleaved by a cellular protease to yield theexternal 120-kDa envelope glycoprotein (gp120) and the transmembrane 41k-Da envelope glycoprotein (gp41). Gp120 and gp41 remain associated andare displayed on the viral particles and the surface of HIV-infectedcells. Gp120 binds to the CD4 receptor present on the surface of helperT-lymphocytes, macrophages and other target cells. After gp120 binds toCD4, gp41 mediates the fusion event responsible for virus entry.

[0004] Infection begins when gp120 on the viral particle binds to theCD4 receptor on the surface of T4 lymphocytes or other target cells. Thebound virus merges with the target cell and reverse transcribes its RNAgenome into the double-stranded DNA of the cell. The viral DNA isincorporated into the genetic material in the cell's nucleus, where theviral DNA directs the production of new viral RNA, viral proteins, andnew virus particles. The new particles bud from the target cell membraneand infect other cells.

[0005] Destruction of T4 lymphocytes, which are critical to immunedefense, is a major cause of the progressive immune dysfunction that isthe hallmark of HIV infection. The loss of target cells seriouslyimpairs the body's ability to fight most invaders, but it has aparticularly severe impact on the defenses against viruses, fungi,parasites and certain bacteria, including mycobacteria.

[0006] HIV-1 kills the cells it infects by replicating, budding fromthem and damaging-the cell membrane. HIV-1 may kill target cellsindirectly by means of the viral gp120 that is displayed on an infectedcell's surface. Since the CD4 receptor on T cells has a strong affinityfor gp120, healthy cells expressing CD4 receptor can bind to gp120 andfuse with infected cells to form a syncytium.

[0007] HIV-1 can also elicit normal cellular immune defenses againstinfected cells. With or without the help of antibodies, cytotoxicdefensive cells can destroy an infected cell that displays viralproteins on its surface. Finally, free gag and gp120 protein maycirculate in the blood of individuals infected with HIV-1. The freegp120 protein may bind to the CD4 receptor of uninfected cells, makingthem appear to be infected and evoking an immune response.

[0008] Infection with HIV-1 is almost always fatal, and at present thereare no cures for HIV-1 infection. Effective vaccines for-prevention ofHIV-1 infection are not yet available. Because of the danger ofreversion or infection, live attenuated virus probably cannot be used asa vaccine. Most subunit vaccine approaches have not been successful atpreventing HIV infection. Treatments for HIV-1 infection, whileprolonging the lives of some infected persons, have serious sideeffects. There is thus a great need for effective treatments andvaccines to combat this lethal infection.

[0009] Vaccination is an effective form of disease prevention and hasproven successful against several types of viral infection. Determiningways to present HIV-1 antigens to the human immune system in order toevoke protective humoral and cellular immunity, is a difficult task. Todate, attempts to generate an effective HIV vaccine have not beensuccessful. In AIDS patients, free virus is present in low levels only.Transmission of HIV-1 is enhanced by cell-to-cell interaction via fusionand syncytia formation. Hence, antibodies generated against free virusor viral subunits are generally ineffective in eliminatingvirus-infected cells.

[0010] Vaccines exploit the body's ability to “remember” an antigen.After first encounters with a given antigen the immune system generatescells that retain an immunological memory of the antigen for anindividual's lifetime. Subsequent exposure to the antigen stimulates theimmune response and results in elimination or inactivation of thepathogen.

[0011] The immune system deals with pathogens in two ways: by humoraland by cell-mediated responses. In the humoral response lymphocytesgenerate specific antibodies that bind to the antigen thus inactivatingthe pathogen. The cell-mediated response involves cytotoxic and helper Tlymphocytes that specifically attack and destroy infected cells.

[0012] Vaccine development with HIV-1 virus presents problems becauseHIV-1 infects some of the same cells the vaccine needs to activate inthe immune system (i.e., T4 lymphocytes). It would be advantageous todevelop a vaccine which inactivates HIV before impairment of the immunesystem occurs. A particularly suitable type of HIV vaccine wouldgenerate an anti-HIV immune response which recognizes HIV variants andwhich works in HIV-positive individuals who are at the beginning oftheir infection.

[0013] A major challenge to the development of vaccines against viruses,particularly those with a high rate of mutation such as the humanimmunodeficiency virus, against which elicitation of neutralizing andprotective immune responses is desirable, is the diversity of the viralenvelope proteins among different viral isolates or strains. Becausecytotoxic T-lymphocytes (CTLs) in both mice and humans are capable ofrecognizing epitopes derived from conserved internal viral proteins, andare thought to be important in the immune response against viruses,efforts have been directed towards the development of CTL vaccinescapable of providing heterologous protection against different viralstrains.

[0014] It is known that CD8+ CTLs kill virally-infected cells when theirT cell receptors recognize viral peptides associated with MHC class Imolecules. The viral peptides are derived from endogenously synthesizedviral proteins, regardless of the protein's location or function withinthe virus. Thus, by recognition of epitopes from conserved viralproteins, CTLs may provide cross-strain protection. Peptides capable ofassociating with MHC class I for CTL recognition originate from proteinsthat are present in or pass through the cytoplasm or endoplasmicreticulum. In general, exogenous proteins, which enter the endosomalprocessing pathway (as in the case of antigens presented by MHC class IImolecules), are not effective at generating CD8+ CTL responses.

[0015] Most efforts to generate C&L responses have used replicatingvectors to produce the protein antigen within the cell or have focusedupon the introduction of peptides into the cytosol. These approacheshave limitations that may reduce their utility as vaccines. Retroviralvectors have restrictions on the size and structure of polypeptides thatcan be expressed as fusion proteins while maintaining the ability of therecombinant virus to replicate, and the effectiveness of vectors such asvaccinia for subsequent immunizations may be compromised by immuneresponses against the vectors themselves. Also, viral vectors andmodified pathogens have inherent risks that may hinder their use inhumans. Furthermore, the selection of peptide epitopes to be presentedis dependent upon the structure of an individual's MHC antigens and,therefore, peptide vaccines may have limited effectiveness due to thediversity of MHC haplotypes in outbred populations.

[0016] Benvenisty, N., and Reshef, L. [PNAS 83, 9551-9555, (1986)]showed that CaCl₂-precipitated DNA introduced into miceintraperitoneally (i.p.), intravenously (i.v.) or intramuscularly (i.m.)could be expressed. The i.m. injection of DNA expression vectors withoutCaCl₂ treatment in mice resulted in the uptake of DNA by the musclecells and expression of the protein encoded by the DNA. The plasmidswere maintained episomally and did not replicate. Subsequently,persistent expression has been observed after i.m. injection in skeletalmuscle of rats, fish and primates, and cardiac muscle of rats. Thetechnique of using nucleic acids as therapeutic agents was reported inWO90/11092 (4 Oct. 1990), in which naked polynucleotides were used tovaccinate vertebrates.

[0017] It is not necessary for the success of the method thatimmunization be intramuscular. The introduction of gold,microprojectiles coated with DNA encoding human growth hormone (HGH)into the skin of mice resulted in production of anti-HGH antibodies inthe mice. A jet injector has been used to transfect skin, muscle, fat,and mammary tissues of living animals. Various methods for introducingnucleic have been reviewed. Intravenous injection of a DNA:cationicliposome complex in mice was shown by Zhu et al., [Science 261:209-211(9 Jul. 1993) to result in systemic expression of a cloned transgene.Ulmer et al., [Science 259:1745-1749, (1993)] reported on theheterologous protection against influenza virus infection byintramuscular injection of DNA encoding influenza virus proteins.

[0018] The need for specific therapeutic and prophylactic agents capableof eliciting desired immune responses against pathogens and tumorantigens is met by the instant invention. Of particular importance inthis therapeutic approach is the ability to induce T-cell immuneresponses which can prevent infections or disease caused even by virusstrains which are heterologous to the strain from which the antigen genewas obtained. This is of particular concern when dealing with HIV asthis virus has been recognized to mutate rapidly and many virulentisolates have been identified [see, for example, LaRosa et al., Science249:932-935 (1990), identifying 245 separate HIV isolates]. In responseto this recognized diversity, researchers have attempted to generateCTLs based on peptide immunization. Thus, Takahashi et al., [Science255:333-336 (1992)] reported on the induction of broadly cross-reactivecytotoxic T cells recognizing an HIV envelope (gp160). determinant.However, those workers recognized the difficulty in, achieving a trulycross-reactive CTL response and suggested that there is a dichotomybetween the priming or restimulation of T cells, which is verystringent, and the elicitation of effector function, includingcytotoxicity, from already stimulated CTLs.

[0019] Wang et al. reported on elicitation of immune responses in miceagainst HIV by intramuscular inoculation with a cloned, genomic(unspliced) HIV gene. However, the level of immune responses achieved inthese studies was very low. In addition, the Wang et al., DNA constructutilized an essentially genomic piece of HIV encoding contiguousTat/rev-gp160-Tat/rev coding sequences. As is described in detail below,this is a suboptimal-system for obtaining high-level expression of thegp160. It also is potentially dangerous because expression of Tatcontributes to the progression of Kaposi's Sarcoma.

[0020] WO 93/17706 describes a method for vaccinating, an animal againsta virus, wherein carrier particles were coated with a gene construct andthe coated particles are accelerated into cells of an animal. In regardto HIV, essentially the entire genome, minus the long terminal repeats,was proposed to be used. That method represents substantial risks forrecipients. It is generally believed that constructs of HIV shouldcontain less than about 50% of the HIV genome to ensure safety of thevaccine; this ensures that enzymatic moieties and viral regulatoryproteins, many of which have unknown or poorly understood functions havebeen eliminated. Thus, a number of problems remain if a useful human HIVvaccine is to emerge from the gene-delivery technology.

[0021] The instant invention contemplates any of the known methods forintroducing polynucleotides into living tissue to induce expression ofproteins. However, this invention provides a novel immunogen forintroducing HIV and other proteins into the antigen processing pathwayto efficiently generate HIV-specific CTLs and antibodies. Thepharmaceutical is effective as a vaccine to induce both cellular andhumoral anti-HIV and HIV neutralizing immune responses. In the instantinvention, the problems noted above are addressed and solved by theprovision of polynucleotide immunogens which, when introduced into ananimal, direct the efficient expression of HIV proteins and epitopeswithout the attendant risks associated with those methods. The immuneresponses thus generated are effective at recognizing HIV, at inhibitingreplication of HIV, at identifying and killing cells infected with HIV,and are cross-reactive against many HIV strains.

[0022] The codon pairings of organisms are highly nonrandom, and differfrom organism to organism. This information is used to construct andexpress altered or synthetic genes having desired levels oftranslational efficiency, to determine which regions in a genome areprotein coding regions, to introduce translational pause sites intoheterologous genes, and to ascertain relationship or ancestral origin ofnucleotide sequences.

[0023] The expression of foreign heterologous genes in transformedorganisms is now commonplace. A large number of mammalian genes,including, for example, murine and human genes, have been successfullyinserted into single celled organisms. Standard techniques in thisregard include introduction of the foreign gene to be expressed into avector such as a plasmid or a phage and utilizing that vector to insertthe gene into an organism. The native promoters for such genes arecommonly replaced with strong promoters compatible with the host intowhich the gene is inserted. Protein sequencing machinery permitselucidation of the amino acid sequences of even minute quantities ofnative protein. From these amino acid sequences, DNA sequences codingfor those proteins can be inferred. DNA synthesis is also a rapidlydeveloping art, and synthetic genes corresponding to those inferred DNAsequences can be readily constructed.

[0024] Despite the burgeoning knowledge of expression systems andrecombinant DNA, significant obstacles remain when one attempts toexpress a foreign or synthetic gene in an organism. Many native, activeproteins, for example, are glycosylated in a manner different from thatwhich occurs when they are expressed in a foreign host. For this,reason, eukaryotic hosts such as yeast may be preferred to bacterialhosts for expressing many mammalian genes. The glycosylation problem isthe subject of continuing research.

[0025] Another problem is more poorly understood. Often translation of asynthetic gene, even when coupled with a strong promoter, proceeds muchless efficiently than would be expected. The same is frequently true ofexogenous genes foreign to the expression organism. Even when the geneis transcribed in a sufficiently efficient manner that recoverablequantities of the translation product are produced, the protein is ofteninactive or otherwise different in properties from the native protein.

[0026] It is recognized that the latter problem is commonly due todifferences in protein folding in various organisms. The solution tothis problem has been elusive, and the mechanisms controlling proteinfolding are poorly understood.

[0027] The problems related to translational efficiency are believed tobe related to codon context effects. The protein coding regions of genesin all organisms are subject to a wide variety of functionalconstraints, some of which depend on the requirement for encoding aproperly functioning protein, as well as appropriate translational startand stop signals. However, several features of protein coding regionshave been discerned which are not readily understood in terms of theseconstraints. Two important classes of such features are those involvingcodon usage and codon context.

[0028] It is known that codon utilization is highly biased and variesconsiderably between different organisms. Codon usage patterns have beenshown to be related to the relative abundance of tRNA isoacceptors.Genes encoding proteins of high versus low abundance show differences intheir codon preferences. The possibility that biases in codon usagealter peptide elongation rates has been widely discussed. Whiledifferences in codon use are associated with differences in translationrates, direct effects of codon choice on translation have been difficultto demonstrate. Other proposed constraints on codon usage patternsinclude maximizing the fidelity of translation and optimizing thekinetic efficiency of protein synthesis.

[0029] Apart from the non-random use of codons, considerable evidencehas accumulated that codon/anticodon recognition is influenced bysequences outside the codon itself, a phenomenon termed “codon context.”There exists a strong influence of nearby nucleotides on the efficiencyof suppression of nonsense codons as well as missense codons. Clearly,the abundance of suppresser activity in natural bacterial populations,as well as the use of “terminiation” codons to encode selenocysteine andphosphoserine require that termination be context-dependent. Similarcontext effects have been shown to influence the fidelity oftranslation, as well as the efficiency of translation initiation.

[0030] Statistical analyses of protein coding regions of E. colidemonstrate another manifestation of “codon context.” The presence of aparticular codon at one position strongly influences the frequency ofoccurrence of certain nucleotides in neighboring codons, and thesecontext constraints differ markedly for genes expressed at high versuslow levels. Although the context effect has been recognized, thepredictive value of the statistical rules relating to preferrednucleotides adjacent to codons is relatively low. This has limited theutility of such nucleotide preference data for selecting codons toeffect desired -levels of translational efficiency.

[0031] The advent of automated nucleotide sequencing equipment has madeavailable large quantities of sequence data for a wide variety oforganisms. Understanding those data presents substantial difficulties.For example, it is important to identify the coding regions of thegenome in order to relate the genetic sequence data to proteinsequences. In addition, the ancestry of the genome of certain organismsis of substantial interest. It is known that genomes of some organismsare of mixed ancestry. Some sequences that are viral in origin are nowstably incorporated into the genome of eukaryotic organisms. The viralsequences themselves may have originated in another substantiallyunrelated species. An understanding of the ancestry of a gene can beimportant in drawing proper analogies between related genes and theirtranslation products in other organisms.

[0032] There is a need for a better understanding of codon contexteffects on translation, and for a method for determining the appropriatecodons for any desired translational effect. There is also a need for amethod for identifying coding regions of the genome from nucleotidesequence data. There is also a need for a method for controlling proteinfolding and for insuring that a foreign gene will fold appropriatelywhen expressed in a host. Genes altered or constructed in accordancewith desired translational efficiencies would be of significant worth.

[0033] Another aspect of the practice of recombinant DNA techniques forthe expression by microorganisms of proteins of industrial andpharmaceutical interest is the phenomenon of “codon preference”. Whileit was earlier noted that the existing machinery for gene expression ingenetically transformed host cells will “operate” to construct a givendesired product, levels of expression attained in a microorganism can besubject to wide variation, depending in part on specific alternativeforms of the amino acid-specifying genetic code present in an insertedexogenous gene. A “triplet” codon of four possible nucleotide bases canexist in 64 variant forms. That these forms provide the message for only20 different amino acids (as well as transcription initiation andtermination) means that some amino acids can be coded for by more thanone codon. Indeed, some amino acids have as many as six “redundant”,alternative codons while some others have a single, required codon. Forreasons not completely understood, alternative codons are not at alluniformly present in the endogenous DNA of differing types of cells andthere appears to exist variable natural hierarchy or “preference” forcertain codons in certain types of cells.

[0034] As one example, the amino acid leucine is specified by any of sixDNA codons including CTA, CTC, CTG, CTT, ITA, and TTG (which correspond,respectively, to the mRNA codons, CUA, CUC, CUG, CUU, UUA and UUG).Exhaustive analysis of genome codon frequencies for microorganisms hasrevealed endogenous DNA of E. coli most commonly contains the CTGleucine-specifying codon, while the DNA of yeasts and slime molds mostcommonly includes a TTA leucine-specifying codon. In view of thishierarchy, it is generally held that the likelihood of obtaining highlevels of expression of a leucine-rich polypeptide by an E. coli hostwill depend to some extent on the frequency of codon use. For example, agene rich in TTA codons will in all probability be poorly expressed inE. coli, whereas a CTG rich gene will probably highly express thepolypeptide. Similarly, when yeast cells are the projectedtransformation host cells for expression of a leucine-rich polypeptide,a preferred codon for use in an inserted DNA would be TTA.

[0035] The implications of codon preference phenomena on recombinant DNAtechniques are manifest, and the phenomenon may serve to explain manyprior failures to achieve high expression levels of exogenous genes insuccessfully transformed host organisms-a less “preferred” codon may berepeatedly present in the inserted gene and the host cell machinery forexpression may not operate as efficiently. This phenomenon suggests thatsynthetic genes which have been designed to include a projected hostcell's preferred codons provide a preferred form of foreign geneticmaterial for practice of recombinant DNA techniques.

[0036] The diversity of function that typifies eukaryotic cells dependsupon the structural differentiation of their membrane boundaries. Togenerate and maintain these structures, proteins must be transportedfrom their site of synthesis in the endoplasmic reticulum topredetermined destinations throughout the cell. This requires that thetrafficking proteins display sorting signals that are recognized by themolecular machinery responsible for route selection located at theaccess points to the main trafficking pathways. Sorting decisions formost proteins need to be made only once as they traverse theirbiosynthetic pathways since their final destination, the cellularlocation at which they perform their function, becomes their permanentresidence.

[0037] Maintenance of intracellular integrity depends in part on theselective sorting and accurate transport of proteins to their correctdestinations. Over the past few years the dissection of the molecularmachinery for targeting and localization of proteins has been studiedvigorously. Defined sequence motifs have been identified on proteinswhich can act as ‘address labels’. Leader or signal peptides such asthat from the tissue-specific plasminogen activator protein, tPA, serveto transport a protein into the cellular secretory pathway through theendoplasmic reticulum and golgi apparatus. A number of sorting signalshave been found associated with the cytoplasmic domains of membraneproteins such as di-Leucine amino acid motifs or tyrosine-basedsequences that can direct proteins to lysosomal compartments. For HIV,transport and extrusion from the cell of viral particles depend uponmyristoylation of glycine residue number two at the amino terminus ofgag.

SUMMARY OF THE INVENTION

[0038] Synthetic DNA molecules encoding HIV gag and modifications of HIVgag are provided. The codons of the synthetic molecules include theprojected host cell's preferred codons. The synthetic molecules providepreferred forms of foreign genetic material. The synthetic molecules maybe used as a polynucleotide vaccine which provides effectiveimmunoprophylaxis against HIV infection through neutralizing antibodyand cell-mediated immunity. This invention provides polynucleotideswhich, when directly introduced into a vertebrate in vivo includingmammals such as primates and humans, induce the expression of encodedproteins within the animal.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039]FIG. 1 shows the relative expression of gag and tPA gag in 293cell line transfectants after transfection with HIV gag DNA.

[0040]FIG. 2 shows optimized HIV gag and tPA-gag DNA vaccine mediatedserum anti-gag responses in mice.

[0041]FIG. 3 shows anti-gag CTL responses of splenocytes obtained frommice following vaccination with optimized HIV gag or tPA-gag DNA.

[0042]FIG. 4 shows gag antigen-specific cytokine secretion ofsplenocytes obtained from mice following vaccination with optimized HIVgag or tPA-gag DNA.

[0043]FIG. 5 shows anti-HIV gag CTL from mice vaccinated with HIV p55gag DNAs.

DETAILED DESCRIPTION OF THE INVENTION

[0044] Synthetic DNA molecules encoding HIV gag and synthetic DNAmolecules encoding modified forms of HIV gag are provided. The codons ofthe synthetic molecules are designed so as to use the codons preferredby the projected host cell. The synthetic molecules may be used as apolynucleotide vaccine which provides effective immunoprophylaxisagainst HIV infection through neutralizing antibody and cell-mediatedimmunity. The synthetic molecules may be used as an immunogeniccomposition. This invention provides polynucleotides which, whendirectly introduced into a vertebrate in vivo, including mammals such asprimates and humans, induce the expression of encoded proteins withinthe animal.

[0045] As used herein, a polynucleotide is a nucleic acid which containsessential regulatory elements such that upon introduction into a living,vertebrate cell, it is able to direct the cellular machinery to producetranslation products encoded by the genes comprising the polynucleotide.In one embodiment of the invention, the polynucleotide is apolydeoiyribonucleic acid comprising at least one HIV gene operativelylinked to a transcriptional promoter. In another embodiment of theinvention, the polynucleotide vaccine (PNV) comprises polyribonucleicacid encoding at least one HIV gene which is amenable to translation bythe eukaryotic cellular machinery (ribosomes, tRNAs, and othertranslation factors). Where the protein encoded by the polynucleotide isone which does not normally occur in that animal except in pathologicalconditions, (i.e., a heterologous protein) such as proteins associatedwith human immunodeficiency virus, (HIV), the etiologic agent ofacquired immune deficiency syndrome, (AIDS), the animals' immune systemis activated to launch a protective immune response. Because theseexogenous proteins are produced by the animals' tissues, the expressedproteins are processed by the major histocompatibility system, MHC, in afashion analogous to when an actual infection with the related organism(HIV) occurs. The result, as shown in this disclosure, is induction ofimmune responses against the cognate pathogen.

[0046] Accordingly, the instant inventors have prepared nucleic acidswhich, when introduced into the biological systemminduce the expressionof HIV proteins and epitopes. The induced antibody response is bothspecific for the expressed HIV protein, and neutralizes HIV. Inaddition, cytotoric T-lymphocytes which specifically recognize anddestroy HIV infected cells are induced.

[0047] The instant invention provides a method for using apolynucleotide which, upon introduction into mammalian tissue, inducesthe expression in a single cell, in vivo, of discrete gene products. Theinstant invention provides a different solution which does not requiremultiple manipulations of rev dependent HIV genes to obtainrev-independent genes. The rev-independent expression system describedherein is useful in its own right and is a system for demonstrating theexpression in a single cell in vivo of a single desired gene-product.

[0048] Because many of the applications of the instant invention applyto anti-viral vaccination, the polynucleotides are frequently referredto as a polynucleotide vaccine, or PNV. This is not to say thatadditional utilities of these polynucleotides, in immune stimulation andin anti-tumor therapeutics, are considered to be outside the scope ofthe invention.

[0049] In one embodiment of this invention, a gene encoding an HIV geneproduct is incorporated in an expression vector. The vector contains atranscriptional promoter recognized by an eukaryotic RNA polymerase, anda transcriptional terminator at the end of the HIV gene coding sequence.In a preferred embodiment, the promoter is the cytomegalovirus promoterwith the intron A sequence (CMV-intA), although those skilled in the artwill recognize that any of a number of other known promoters such as thestrong immunoglobulin, or other eukaryotic gene promoters may be used. Apreferred transcriptional terminator is the bovine growth hormoneterminator. The combination of CMVintA-BGH terminator is particularlypreferred.

[0050] To assist in preparation of the polynucleotides in prokaryoticcells, an antibiotic resistance marker is also preferably included inthe expression vector under transcriptional control of a prokaryoticpromoter so that expression of the antibiotic does not occur ineukaryotic cells. Ampicillin resistance genes, neomycin resistance genesand other pharmaceutically acceptable antibiotic resistance markers maybe used. To aid in the high level production of the polynucleotide byfermentation in prokaryotic organisms, it is advantageous for the vectorto contain a prokaryotic origin of replication and be of high copynumber. A number of commercially available prokaryotic cloning vectorsprovide these benefits. It is desirable to remove non-essential DNAsequences. It is also desirable that the vectors not be able toreplicate in eukaryotic cells. This minimizes the risk of integration ofpolynucleotide vaccine sequences into the recipients' genome.Tissue-specific promoters or enhancers may be used whenever it isdesirable to limit expression of the polynucleotide to a particulartissue type.

[0051] In one embodiment, the expression vector pnRSV is used, whereinthe Rous Sarcoma Virus (RSV) long terminal repeat (LTR) is used as thepromoter. In another embodiment, V1, a mutated pBR322 vector into whichthe CMV promoter and the BGH transcriptional terminator were cloned isused. In another embodiment, the elements of V1 and pUC19 have beencombined to produce an expression vector named V1J. Into V1J or anotherdesirable expression vector is cloned an HIV gene, such as gp120, gp41,gp160, gag, pol, env, or any other HIV gene which can induce anti-HIVimmune responses. In another embodiment, the ampicillin resistance geneis removed from V1J and replaced with a neomycin resistance gene, togenerate V1J-neo into different HIV genes have been cloned for useaccording to this invention. In another embodiment, the vector is V1Jns,which is the same as V1Jneo except that a unique Sfi1 restriction sitehas been engineered into the single Kpn1site at position 2114 ofV1J-neo. The incidence of Sfi1 sites in human genomic DNA is very low(approximately 1 site per 100,000 bases). Thus, this vector allowscareful monitoring for expression vector integration into host DNA,simply by Sfi1 digestion of extracted genoraic DNA. In a furtherrefinement, the vector is V1R. In this vector, as much non-essential DNAas possible was “trimmed” from the vector to produce a highly compactvector. This vector is a derivative of V1Jns. This vector allows largerinserts to be used, with less concern that undesirable sequences areencoded and optimizes uptake by cells.

[0052] One embodiment of this invention incorporates genes encoding HIVgag from laboratory adapted strains of HIV such as IIIB or CAM-1. Thoseskilled in the art will recognize that the use of genes from otherstrains of HIV-1 or HIV-2 strains having analogous function to the genesfrom HIV-1 would be expected to generate immune responses analogous tothose described herein for HIV-1 constructs. The cloning andmanipulation methods for obtaining these genes are known to thoseskilled in the art.

[0053] Sequences for many genes of many HIV strains are now publiclyavailable on GENBANK and such primary, field isolates of HIV areavailable from the National Institute of Allergy and Infectious Diseases(NIAID) which has contracted with Quality Biological, Inc., [7581Lindbergh Drive, Gaithersburg, Md. 20879] to make these strainsavailable. Such strains are also available from the World HealthOrganization (WHO) [Network for HIV Isolation and Characterization,Vaccine Development Unit, Office of Research, Global Programme on AIDS,CH-1211 Geneva 27, Switzerland]. From this work those skilled in the artwill recognize that one of the utilities of the instant invention is toprovide a system for in vivo as well as in vitro testing and analysis sothat a correlation of HIV sequence diversity with serology of HIVneutralization, as well as other parameters can be made. Incorporationof genes from primary isolates of HIV strains provides an immunogenwhich induces immune responses against clinical isolates of the virusand thus meets a need as yet unmet in the field. Furthermore, as thevirulent isolates-change, the immunogen may be modified to reflect newsequences as necessary.

[0054] To keep the terminology consistent, the following convention isfollowed herein for describing polynucleotide immunogen constructs:“Vector name-HIV strain-gene-additional elements”. The additionalelements that are added to the construct are described in further detailbelow. As the etiologic strain of the virus changes, the precise genewhich is optimal for incorporation in the pharmaceutical may be changed.However, as is demonstrated below, because CTL responses are inducedwhich are capable of protecting against heterologous strains, the strainvariability is less critical in the immunogen and vaccines of thisinvention, as compared with the whole virus or subunit polypeptide basedvaccines. In addition, because the pharmaceutical is easily manipulatedto insert a new gene, this is an adjustment which is easily made by thestandard techniques of molecular biology.

[0055] The term “promoter” as used herein refers to a recognition siteon a DNA strand to which the RNA polymerase binds. The promoter forms aninitiation complex with RNA polymerase to initiate and drivetranscriptional activity. The complex can be modified by activatingsequences termed “enhancers” or inhibiting sequences termed “silencers.”

[0056] The term “leader” as used herein refers to a DNA sequence at the5′ end of a structural gene which is transcribed along with the gene.The leader usually results in the protein having an N-terminal peptideextension sometimes called a pro-sequence. For proteins destined foreither secretion to the extracellular medium or a membrane, this signalsequence, which is generally hydrophobic, directs the protein intoendoplasmic reticulum from which it is discharged to the appropriatedestination.

[0057] The term “intron” as used herein refers to a section of DNAoccurring in the middle of a gene which does not code for an amino acidin the gene product. The precursor RNA of the intron is excised and istherefore not transcribed into mRNA nor translated into protein.

[0058] The term “cassette” refers to the sequence of the presentinvention which contains the nucleic acid sequence which is to beexpressed. The cassette is similar in concept to a cassette tape. Eachcassette will have its own sequence. Thus by interchanging the cassettethe vector will express a different sequence. Because of therestrictions sites at the 5′ and 3′ ends, the cassette can be easilyinserted, removed or replaced with another cassette.

[0059] The term “3′ untranslated region” or “3′ UTR” refers to thesequence at the 3′ end of a structural gene which is usually transcribedwith the gene. This 3′ UTR region usually contains the poly A sequence.Although the 3′ UTR is transcribed from the DNA it is excised beforetranslation into the protein.

[0060] The term “Non-Coding Region” or “NCR” refers to the region whichis contiguous to the 3′ UTR region of the structural gene. The NCRregion contains a transcriptional termination signal.

[0061] The term “restriction site” refers to a sequence specificcleavage site of restriction endonucleases.

[0062] The term “vector” refers to some means by which DNA fragments canbe introduced into a host organism or host tissue. There are varioustypes of vectors including plasmid, bacteriophages and cosmids.

[0063] The term “effective amount” means sufficient PNV is injected toproduce the adequate levels of the polypeptide. One skilled in the artrecognizes that this level may vary.

[0064] To provide a description of the instant invention, the followingbackground on HIV is provided. The human immunodeficiency virus has aribonucleic acid (RNA) genome. This RNA genome must be reversetranscribed according to methods known in the art in order to produce acDNA copy for cloning and manipulation according to the methods taughtherein. At each end of the genome is a long terminal repeat which actsas a promoter. Between these termini, the genome encodes, in variousreading frames, gag-pol-env as the major gene products: gag is the groupspecific antigen; pol is the reverse transcriptase, or polymerase; also,encoded by this region, in an alternate reading frame, is the viralprotease which is responsible for post-translational processing, forexample, of gp160 into gp120 and gp41; env is the envelope protein; vifis the virion infectivity factor; rev is the regulator of virion proteinexpression; nef is the negative regulatory factor; vpu is the virionproductivity factor “u”; tat is the trans-activator of transcription;vpr is the viral protein r. The function of each of these elements hasbeen described.

[0065] In one embodiment of this invention, a gene encoding an HIV gagprotein is directly linked to a transcriptional promoter. However,expression of gag is repressed in the absence of rev due to non-exportfrom the nucleus of non-spliced genes. For an understanding of thissystem, the life cycle of HIV must be described in further detail.

[0066] In the-life cycle of HIV, upon infection of a host cell, HIV RNAgenome is reverse-transcribed into a proviral DNA which integrates intohost genomic DNA as a single transcriptional unit.

[0067] The LTR provides the promoter which transcribes HIV genes fromthe 5′ to 3′ direction (gag, pol, env), to form an unspliced transcriptof the entire genome. The unspliced transcript functions as the mRNAfrom which gag and pol are translated, while limited splicing must occurfor translation of env encoded genes. For the regulatory gene productrev to be expressed, more than one splicing event must occur because inthe genomic setting, rev and env, as is shown in FIG. 1, overlap. Inorder for transcription of env to occur, rev transcription must stop,and vice versa. In addition, the presence of rev is required for exportof unspliced RNA from the nucleus. For rev to function in this manner,however, a rev responsive element (RRE) must be present on thetranscript [Malim et al., Nature 33:254-257 (1989)].

[0068] In the polynucleotide vaccine of this invention, the obligatorysplicing of certain HIV genes is eliminated by providing fully splicedgenes (i.e.: the provision of a complete open reading frame for thedesired gene product without the need for switches in the reading frameor elimination of noncoding regions; those of ordinary skill in the artwould recognize that when splicing a particular gene, there is somelatitude in the precise sequence that results; however so long as afunctional coding sequence is obtained, this is acceptable). Thus, inone embodiment, the entire coding sequence for gag is spliced such thatno intermittent expression of each gene product is required.

[0069] The dual humoral and cellular immune responses generatedaccording to this invention are particularly significant to inhibitingHIV infection, given the propensity of HIV to mutate within thepopulation, as well as in infected individuals. In order to formulate aneffective protective vaccine for HIV it is desirable to generate both amultivalent antibody response for example to gp160 (env is approximately80% conserved across various HIV-1, code B strains, which are theprevalent strains in US human populations), the principal neutralizationtarget on HIV, as well as cytotoric T cells reactive to the conservedportions of gp160 and, internal viral proteins encoded by gag. We havemade an HIV vaccine comprising gp160 genes selected from commonlaboratory strains; from predominant, primary viral isolates foundwithin the infected population; from mutated gp160₆ designed to unmaskcross-strain, neutralizing antibody epitopes; and from otherrepresentative HIV genes such as the gag and pol genes (˜95% conservedacross HIV isolates.

[0070] Virtually all HIV seropositive patients who have not advancedtowards an immunodeficient state harbor anti-gag CTLs while about 60% ofthese patients show cross-strain, gp160-specific CTLs. The amount of HIVspecific CTLs found in infected individuals that have progressed on tothe disease state known as AIDS, however, is much lower, demonstratingthe-significance of our findings that we can induce cross-strain CTLresponses.

[0071] Immune responses induced by our env and gag polynucleotidevaccine constructs are demonstrated in mice and primates. Monitoringantibody production to env in mice allows configuration that a givenconstruct is suitably immunogenic, i.e., a high proportion of vaccinatedanimals show an antibody response. Mice also provide the most facileanimal model suitable for testing CTL induction by our constructs andare therefore used to evaluate whether a particular construct is able togenerate such activity. Monkeys (African green, rhesus, chimpanzees)provide additional species including primates for antibody evaluation inlarger, non-rodent animals. These species are also preferred to mice forantisera neutralization assays due to high levels of endogenousneutralizing activities against retroviruses observed in mouse sera.These data demonstrate that sufficient immunogenicity is engendered byour vaccines to achieve protection in experiments in achimpanzee/HIV_(IIIB) challenge model based upon known protective levelsof neutralizing antibodies for this system. However, the currentlyemerging and increasingly accepted definition of protection in thescientific community is moving away from so-called “sterilizingimmunity”, which indicates complete protection from HIV infection, toprevention of disease. A number of correlates of this goal includereduced blood viral titer, as measured by PCR, HIV reverse transcriptaseactivity, by infectivity of samples of serum, by ELISA assay of p24 orother HIV antigen concentration in blood, increased CD4⁺ T-cellconcentration, and by extended survival rates [see, for example, Cohen,J., Science 262:1820-1821, 1993, for a discussion of the evolvingdefinition of anti-HIV vaccine efficacy]. The immunogens of the instantinvention also generate neutralizing immune responses against infectious(clinical, primary field) isolates of HIV.

[0072] An ELISA assay is used to determine whether optimized gag vaccinevectors expressing either secreted tPA-gag or native gag are efficaciousfor production of gag-specific antibodies. Initial in vitrocharacterization of gag expression by our vaccination vectors isprovided by immunoblot analysis of optimized gag transfected celllysates. These data confirm and quantitate gag expression using anti-HIVantisera to visualize transfectant cell gag expression.

[0073] Generation of CTL Responses. Viral proteins which are synthesizedwithin cells give rise to MHC I-restricted CTL responses. Each of theseproteins elicits CTL in seropositive patients. Our vaccines also areable to elicit CTL in mice and rhesus monkeys. The immunogenetics ofmouse strains are conducive to such studies, as demonstrated withinfluenza NP, [see Ulmer et al., Science 259:1745-1749, 1993]. Severalepitopes have been defined for the HIV proteins env, rev, nef and gag(Frankel, F. R., et al., J. Immunol. 155, 4775-82 (1995)in Balb/c mice,thus facilitating in vitro CTL culture and cytotoxicity assays.Alternatively, the entire gene encoding gp160, gp120, pol, or gag couldbe used. For additional review on this subject, see for example, (HIVMolecular Immunology Database 1995, Korber et al. eds., Los AlamosNational Laboratory, Los Alamos, N.Mex., U.S.A.).As used herein, T-celleffector function is associated with mature T-cell phenotype, forexample, cytotoxicity, cytokine secretion for B-cell activation, and/orrecruitment or stimulation of macrophages and neutrophils.

[0074] Measurement of T_(H) Activities. Spleen cell cultures derivedfrom vaccinated animals are tested for recall to specific antigens byaddition of either recombinant protein or peptide epitopes. Activationof T cells by such antigens, presented by accompanying splenic antigenpresenting cells, APCs, is monitored by proliferation of these culturesor by cytokine production. The pattern of cytokine production alsoallows classification of T^(H) response as type 1 or type 2. Becausedominant T^(H)2 responses appear to correlate with the exclusion ofcellular immunity in immunocompromised seropositive patients, it ispossible to define the type of response engendered by a given PNV inpatients, permitting manipulation of the resulting immune responses.

[0075] Based upon the above immunologic studies, it is predictable thatour vaccines are effective in vertebrates against challenge by virulentHIV. These studies are accomplished in an HIV_(IIIB)/chimpanzeechallenge model after sufficient vaccination of these animals with a PNVconstruct, or a cocktail of PNV constructs comprised of gp160_(IIIB),gag_(IIIB), nef_(IIIB) and REV_(IIIB). The IIIB strain is useful in thisregard as the chimpanzee titer of lethal doses of this strain has beenestablished. However, the same studies are envisioned using any strainof HIV and the epitopes specific to or heterologous to the given strain.A second vaccination/challenge model, in addition to chimpanzees, is thescid-hu PBL mouse. This model allows testing of the human lymphocyteimmune system and our vaccine with subsequent HIV challenge in a mousehost. This system is advantageous as it is easily adapted to use withany HIV strain and it provides evidence of protection against multiplestrains of primary field isolates of HIV. A third challenge modelutilizes hybrid HIV/SIV viruses (SHIV), some of which have been shown toinfect rhesus monkeys and lead to immunodeficiency disease resulting indeath [see Li, J., et al., J. AIDS 5:639-646, 1992]. Vaccination ofrhesus with our polynucleotide vaccine constructs is protective againstsubsequent challenge with lethal doses of SHIV.

[0076] There are numerous viral and bacterially derived genes which donot utilize codons that are optimized for expression in mammalian cells.Reasons for this include the fact that these microorganisms providetheir own polymerases or provide specific proteins or factors tofacilitate transcription/translation of their gene products. Forbacteria, there may be different relative abundances for specific tRNAs.Clearly in vivo expression of such genes in the context of a DNA vaccineis substantially different.

[0077] Representative construct components include (but are notrestricted to): HIV env, HIV gag, HV pol, HIV rev, HIV vpr, and HIV nef.Genes encoding antigens expressed by pathogens other than HIV, such as,but not limited to, influenza virus nucleoprotein, hemagglutinin,matrix, neuraminidase, and other antigenic proteins; herpes simplexvirus genes; human papillomavirus genes; tuberculosis antigens;hepatitis A, B, or C virus antigens.

[0078] The protective efficacy of polynucleotide HIV immunogens againstsubsequent viral challenge is demonstrated by immunization with thenon-replicating plasmid DNA of this invention. This is advantageoussince no infectious agent is involved, assembly of virus particles isnot required, and determinant selection is permitted. Furthermore,because the sequence of gag and protease and several of the other viralgene products is conserved among various strains of HIV, protectionagainst subsequent challenge by a virulent strain of HIV that ishomologous to, as well as strains heterologous to the strain from whichthe cloned gene is obtained, is enabled.

[0079] The invention offers a means to induce cross-strain, protectiveimmunity without the need for self-replicating agents or adjuvants. Inaddition, immunization with the instant polynucleotides offers a numberof other advantages. This approach to vaccination should be applicableto tumors as well as infectious agents, since the CD8+ CTL response isimportant for both pathophysiological processes [K. Tanaka et al, Annu.Rev. Immunol. 6, 359 (1988)]. Therefore, eliciting an immune responseagainst a protein crucial to the transformation process may be aneffective means of cancer protection or immunotherapy. The generation ofhigh titer antibodies against expressed proteins after injection ofviral protein and human growth hormone DNA suggests that this is afacile and highly effective means of making antibody-based vaccines,either separately or in combination with cytotoic T-lymphocyte vaccinestargeted towards conserved antigens.

[0080] The ease of producing and purifying DNA constructs comparesfavorably with traditional methods of protein purification, thusfacilitating the generation of combination vaccines. Accordingly,multiple constructs, for example encoding gp160, gp120, gp41, gag, orany other HIV gene may be prepared, mixed and co-administered. Becauseprotein expression is maintained following DNA injection, thepersistence of B- and T-cell memory may be enhanced, thereby engenderinglong-lived humoral and cell-mediated immunity.

[0081] Standard techniques of molecular biology for preparing andpurifying DNA constructs enable the preparation of the DNA immunogens ofthis invention. While standard techniques of molecular biology aretherefore sufficient for the production of the products of thisinvention, the specific constructs disclosed herein provide novelpolynucleotide immunogens which surprisingly produce cross-strain andprimary HIV isolate neutralization, a result heretofore unattainablewith standard inactivated whole virus or subunit protein vaccines.

[0082] The amount of expressible DNA or transcribed RNA to be introducedinto a vaccine recipient will depend on the strength of thetranscriptional and translational promoters used and on theimmunogenicity of the expressed gene product. In general, animmunologically or prophylactically effective dose of about 1 ng to 100mg, and preferably about 10 μg to 300 μg is administered directly intomuscle tissue. Subcutaneous injection, intradermal introduction,impression through the skin, and other modes of administration such asintraperitoneal, intravenous, or inhalation delivery are alsocontemplated. It is also contemplated that booster vaccinations are tobe provided. Following vaccination with HIV polynucleotide imnunogen,boosting with HIV protein immunogens such as gp160, gp120, and gag geneproducts is also contemplated. Parenteral administration, such asintravenous, intramuscular, subcutaneous or other means ofadministration of interleukin-12 protein, concurrently with orsubsequent to parenteral introduction of the PNV of this invention isalso advantageous.

[0083] The polynucleotide may be naked, that is, unassociated with anyproteins, adjuvants or other agents which impact on the recipients'immune system. In this case, it is desirable for the polynucleotide tobe in a physiologically acceptable solution, such as, but not limitedto, sterile saline or sterile buffered saline. Alternatively, the DNAmay be associated with liposomes, such as lecithin liposomes or otherliposomes known in the art, as a DNA-liposome mixture, or the DNA may beassociated with an adjuvant known in the art to boost immune responses,such as a protein or other carrier. Agents which assist in the cellularuptake of DNA, such as, but not limited to, calcium ions, may also beused to advantage. These agents are generally referred to herein astransfection facilitating reagents and pharmaceutically acceptablecarriers. Techniques for coating microprojectiles coated withpolynucleotide are known in the art and are also useful in connectionwith this invention.

[0084] The following examples are offered by way of illustration and arenot intended to limit the invention in any manner.

EXAMPLE 1 Heterologous Expression of HIV Late Gene Products

[0085] HIV structural genes such as env and gag require expression ofthe HIV regulatory gene, rev, in order to efficiently producefull-length proteins. We have found that rev-dependent expression of gagyielded low levels of protein and that rev itself may be toxic to cells.Although we achieved relatively high levels of rev-dependent expressionof gp160 in vitro this vaccine elicited low levels of antibodies togp160 following in vivo immunization with rev/gp160 DNA. This may resultfrom known cytotoxic effects of rev as well as increased difficulty inobtaining rev function in myotubules containing hundreds of nuclei (revprotein needs to be in the same nucleus as a rev-dependent transcriptfor gag or env protein expression to occur). However, it has beenpossible to obtain rev-independent expression using selectedmodifications of the env gene.

[0086] In general, our vaccines have utilized primarily HIV (IIIB, MN orCAM-1) env and gag genes for optimization of expression within ourgeneralized vaccination vector, V1Jns, which is comprised of a CMVimmediate-early (IE) promoter, BGH polyadenylation site, and a pUCbackbone. Varying efficiencies, depending upon how large a gene segmentis used (e.g., gp120 vs. gp160), of rev-independent expression may beachieved for env by replacing its native secretory leader peptide withthat from the tissue-specific plasminogen activator (tPA) gene andexpressing the resulting chimeric gene behind the CMVIE promoter withthe CMV intron A

[0087] As stated previously, we consider realization of the followingobjectives to be essential to maximize our chances for success with thisprogram: (1) env-based vectors capable of generating strongerneutralizing antibody responses in primates; (2) gag and env vectorswhich elicit strong T-lymphocyte responses as characterized by CTL andhelper effector functions in primates; (3) use of env and gag genes fromclinically relevant HIV-1 strains in our vaccines and characterizationof the immunologic responses, especially neutralization of primaryisolates, they elicit; (4) demonstration of protection in an animalchallenge model such as chimpanzee/HIV (IIIB) or rhesus/SHIV usingappropriate optimized vaccines; and, (5) determination of the durationof immune responses appropriate to clinical use. Significant progresshas been made on the first three of these objectives and experiments arein progress to determine whether our recent vaccination constructs forgp160 and gag will improve upon these initial results.

EXAMPLE2 Vectors For Vaccine Production

[0088] A. V1Jneo Expression Vector:

[0089] It was necessary to remove the amp^(r) gene used for antibioticselection of bacteria harboring V1J because ampicillin may not be usedin large-scale fermenters. The amp^(r) gene from the pUC backbone of V1Jwas removed by digestion with SspI and Eam1105I restriction enzymes. Theremaining plasmid was purified by agarose gel electrophoresis,blunt-ended with T4 DNA polymerase, and then treated with calfintestinal alkaline phosphatase. The commercially available kan^(r)gene, derived from transposon 903 and contained within the pUC4Kplasmid, was excised using the PstI restriction enzyme, purified byagarose gel electrophoresis, and blunt-ended with T4 DNA polymerase.This fragment was ligated with the V1J backbone and plasmids with thekan^(r) gene in either orientation were derived which were designated asV1Jneo # s 1 and 3. Each of these plasmids was confirmed by restrictionenzyme digestion analysis, DNA sequencing of the junction regions, andwas shown to produce similar quantities of plasmid as V1J. Expression ofheterologous gene products was also comparable to V1J for these V1Jneovectors. We arbitrarily selected V1Jneo#3, referred to as V1Jneohereafter, which contains the kan^(r) gene in the same orientation asthe amp^(r) gene in V1J as the expression construct.

[0090] B. V1Jns Expression Vector:

[0091] An Sfi I site was added to V1Jneo to facilitate integrationstudies. A commercially available 13 base pair Sfi I linker (New EnglandBioLabs) was added at the Kpn I site within the BGH sequence of thevector. V1Jneo was linearized with Kpn I, gel purified, blunted by T4DNA polymerase, and ligated to the blunt Sfi I linker. Clonal isolateswere chosen by restriction mapping and verified by sequencing throughthe linker. The new vector was designated V1Jns. Expression ofheterologous genes in V1Jns (with Sfi I) was comparable to expression ofthe same genes in V1Jneo (with Kpn I).

[0092] C. V1Jns-tPA:

[0093] In order to provide an heterologous leader peptide sequence tosecreted and/or membrane proteins, V1Jn was modified to include thehuman tissue-specific plasminogen activator (tPA) leader. Two syntheticcomplementary oligomers were annealed and then ligated into V1Jn whichhad been BgIII digested. These oligomers have overhanging basescompatible for ligation to BgIII-cleaved sequences. After ligation theupstream BgIII site is destroyed while the downstream BgIII is retainedfor subsequent ligations. Both the junction sites as well as the entiretPA-leader sequence were verified by DNA sequencing. Additionally, inorder to conform with our consensus optimized vector V1Jns (=V1Jneo withan SfiI site), an SfiI restriction site was placed at the KpnI sitewithin the BGH terminator region of V1Jn-tPA by blunting the KpnI sitewith T4 DNA polymerase followed by ligation with an SfiI linker(catalogue #1138, New England Biolabs). This modification was verifiedby restriction digestion and agarose gel electrophoresis.

[0094] D. Vector V1R Preparation:

[0095] In an effort to continue to optimize our basic vaccinationvector, we prepared a derivative of V1Jns which was designated as V1R.The purpose for this vector construction was to obtain a minimum-sizedvaccine vector, i.e., without unnecessary DNA sequences, which stillretained the overall optimized heterologous gene expressioncharacteristics and high plasmid yields that V1J and V1Jns afford. Wedetermined from the literature as well as by experiment that (1) regionswithin the pUC backbone comprising the E. coli origin of replicationcould be removed without affecting plasmid yield from bacteria; (2) the3′-region of the kan^(r) gene following the kanamycin open reading framecould be removed if a bacterial terminator was inserted in its stead;and, (3)˜300 bp from the 3′-half of the BGH terminator could be removedwithout affecting its regulatory function (following the original KpnIrestriction enzyme site within the BGH element).

[0096] V1R was constructed by using PCR to synthesize three segments ofDNA from V1Jns representing the CMVintA promoter/BGH terminator, originof replication, and kanamycin resistance elements, respectively.Restriction enzymes unique for each segment were added to each segmentend using the PCR oligomers: SspI and XhoI for CMVintA/BGH; EcoRV andBamHI for the kan^(r) gene; and, BcII and SaII for the ori^(r). Theseenzyme sites were chosen because they allow directional ligation of eachof the PCR-derived DNA segments with subsequent loss of each site: EcoRVand SspI leave blunt-ended DNAs which are compatible for ligation whileBamHI and BclI leave complementary overhangs as do SalI and XhoI. Afterobtaining these segments by PCR each segment was digested with theappropriate restriction enzymes indicated above and then ligatedtogether in a single reaction mixture containing all three DNA segments.The 5′-end of the ori^(r) was designed to include the T2 rho independentterminator sequence that is normally found in this region so that itcould provide termination information for the kanamycin resistance gene.The ligated product was confirmed by restriction enzyme digestion (>8enzymes) as well as by DNA'sequencing of the ligation junctions. DNAplasmid yields and heterologous expression using viral genes within V1Rappear similar to V1Jns. The net reduction in vector size achieved was1346 bp (V1Jns=4.86 kb; V1R=3.52 kb).

EXAMPLE 3 Design of Synthetic Gene Segments for Increased gag GeneExpression

[0097] Gene segments were converted to sequences having identicaltranslated sequences but with alternative codon usage as defined by R.Lathe in a research article from J. Molec. Biol. Vol. 183, pp. 1-12(1985) entitled “Synthetic Oligonucleotide Probes Deduced from AminoAcid Sequence Data: Theoretical and Practical Considerations”. Themethodology described below to increase of expression of HIV gag genesegments was based on our hypothesis that the known inability to expressthis gene efficiently in mammalian cells is a consequence of the overalltranscript composition. Thus, using alternative codons encoding the sameprotein sequence may remove the constraints on expression of gag. Thespecific codon replacement method employed may be described as follows:

[0098] 1. Identify placement of codons for proper open reading frame.

[0099] 2. Compare wild type codon for observed frequency of use by humangenes.

[0100] 3. If codon is not the most commonly employed, replace it with anoptimal codon for high expression in human cells.

[0101] 4. Repeat this procedure until the entire gene segment has beenreplaced.

[0102] 5. Inspect new gene sequence for undesired sequences generated bythese codon replacements (e.g., “ATTTA” sequences, inadvertent creationof intron splice recognition sites, unwanted restriction enzyme sites,etc.) and substitute codons that eliminate these sequences.

[0103] 6. Assemble synthetic gene segments and test for improvedexpression.

[0104] These methods were used to create the following synthetic genesegments for HIV gag creating a gene comprised entirely of optimal codonusage for expression. While the above procedure provides a summary ofour methodology for designing codon-optimized genes for DNA vaccines, itis understood by one skilled in the art that similar vaccine efficacy orincreased expression of genes may be achieved by minor variations is theprocedure or by minor variations in the sequence.

EXAMPLE 4 I. HIV gag Vaccine Constructs:

[0105] This is a complete HIV-1(CAM1)gag orf comprised of optimalcodons. 1 AGATCTACCA TGGGTGCTAG GGCTTCTGTG CTGTCTGGTG GTGAGCTGGA (SEQ IDNO:1) 51 CAAGTGGGAG AAGATCAGGC TGAGGCCTGG TGGCAAGAAG AAGTACAAGC 101TAAAGCACAT TGTGTGGGCC TCCAGGGAGC TGGAGAGGTT TGCTGTGAAC 151 CCTGGCCTGCTGGAGACCTC TGAGGGGTGC AGGCAGATCC TGGGCCAGCT 201 CCAGCCCTCC CTGCAAACAGGCTCTGAGGA GCTGAGGTCC CTGTACAACA 251 CAGTGGCTAC CCTGTACTGT GTGCACCAGAAGATTGATGT GAAGGACACC 301 AAGGAGGCCC TGGAGAAGAT TGAGGAGGAG CAGAACAAGTCCAAGAAGAA 351 GGCCCAGCAG GCTGCTGCTG GCACAGGCAA CTCCAGCCAG GTGTCCCAGA401 ACTACCCCAT TGTGCAGAAC CTCCAGGGCC AGATGGTGCA CCAGGCCATC 451TCCCCCCGGA CCCTGAATGC CTGGGTGAAG GTGGTGGAGG AGAAGGCCTT 501 CTCCCCTGAGGTGATCCCCA TGTTCTCTGC CCTGTCTGAG GGTGCCACCC 551 CCCAGGACCT GAACACCATGCTGAACACAG TGGGGGGCCA TCAGGCTGCC 601 ATGCAGATGC TGAAGGAGAC CATCAATGAGGAGGCTGCTG AGTGGGACAG 651 GCTGCATCCT GTGCACGCTG GCCCCATTGC CCCCGGCCAGATGAGGGAGC 701 CCAGGGGCTC TGACATTGCT GGCACCACCT CCACCCTCCA GGAGCAGATT751 GGCTGGATGA CCAACAACCC CCCCATCCCT GTGGGGGAAA TCTACAAGAG 801GTGGATCATC CTGGGCCTGA ACAAGATTGT GAGGATGTAC TCCCCCACCT 851 CCATCCTGGACATCAGGCAG GGCCCCAAGG AGCCCTTCAG GGACTATGTG 901 GACAGGTTCT ACAAGACCCTGAGGGCTGAG CAGGCCTCCC AGGAGGTGAA 951 GAACTGGATG ACAGAGACCC TGCTGGTGCAGAATGCCAAC CCTGACTGCA 1001 AGACCATCCT GAAGGCCCTG GGCCCTGCTG CCACCCTGGAGGAGATGATG 1051 ACAGCCTGCC AGGGGGTGGG GGGCCCTGGT CACAAGGCCA GGGTGCTGGC1101 TGAGGCCATG TCCCAGGTGA CCAACTCCGC CACCATCATG ATGCAGAGGC 1151GCAACTTCAG GAACCAGAGG AAGACAGTGA AGTGCTTCAA CTGTGGCAAG 1201 GTGGGCCACATTGCCAAGAA CTGTAGGGCC CCCAGGAAGA AGGGCTGCTG 1251 GAAGTGTGGC AAGGAGGGCCACCAGATGAA GGACTGCAAT GAGAGGCAGG 1301 CCAACTTCCT GGGCAAAATC TGGCCCTCCCACAAGGGCAG GCCTGGCAAC 1351 TTCCTCCAGT CCAGGCCTGA GCCCACAGCC CCTCCCGAGGAGTCCTTCAG 1401 GTTTGGGGAG GAGAAGACCA CCCCCAGCCA GAAGCAGGAG CCCATTGACA1451 AGGAGCTGTA CCCCCTGGCC TCCCTGAGGT CCCTGTTTGG CAACGACCCC 1501TCCTCCCAGT AAAATAAAGC CCGGGCAGAT CT

EXAMPLE 5 In Vitro gag Vaccine Expression:

[0106] In vitro expression was tested in transfected humanrhabdomyosarcoma (RD) or 293 cells for these constructs. Quantitation ofgag from transfected 293 cells showed that V1Jns-opt-gag andV1Jns-tPA-opt gag vector produced secreted gag.

EXAMPLE6 Assay For HIV-gag Cytotoxic T-Lymphocytes:

[0107] The methods described in this section illustrate the assay asused for vaccinated mice. An essentially similar assay can be used withprimates except that autologous B cell lines must be established for useas target cells for each animal. This can be accomplished for humansusing the Epstein-Barr virus and for rhesus monkey using the herpes Bvirus.

[0108] Peripheral blood mononuclear cells (PBMC) are derived from eitherfreshly drawn blood or spleen using Ficoll-Hypaque centrifugation toseparate erythrocytes from white blood cells. For mice, lymph nodes maybe used as well. Effector CTLs may be prepared from the PBMC either byin vitro culture in IL-2 (20 U/ml) and concanavalin A (2 μg/ml) for 6-12days or by using specific antigen using an equal number of irradiatedantigen presenting cells. Specific antigen can consist of eithersynthetic peptides (9-15 amino acids usually) that are known epitopesfor CTL recognition for the MHC haplotype of the animals used, orvaccinia virus constructs engineered to express appropriate antigen.Target cells may be either syngenic or MHC haplotype-matched cell lineswhich have been treated to present appropriate antigen as described forin vitro stimulation of the CTLs. For Balb/c mice the gag peptide ofPaterson (J. Immunol., 1995), was used at 10 μM concentration torestimulate CTL invitro using irradiated syngenic splenocytes and can beused to sensitize target cells during the cytotoxicity assay at 1-10 μMby incubation at 37° C. for, about two hours prior to the assay. Forthese H-2^(d) MHC haplotype mice, the murine mastocytoma cell line,P815, provides good, target cells. Antigen-sensitized target cells areloaded with Na⁵¹CrO₄, which is released from the interior of the targetcells upon killing by CTL, by incubation of targets for 1-2 hours at37°0 C. (0.2 mCi for ˜5×10⁶ cells) followed by several washings of thetarget cells. CTL populations are mixed with target cells at varyingratios of effectors to targets such as 100:1, 50:1, 25:1, etc., pelletedtogether, and incubated 46 hours at 37° C. before harvest of thesupernatants which are then assayed for release of radioactivity using agamma counter. Cytotoxicity is calculated as a percentage of totalreleasable counts from the target cells (obtained using 0.2% TritonX-100 treatment) from which spontaneous release from target cells hasbeen subtracted.

EXAMPLE 7 Assay for HIV gag Specific Antibodies:

[0109] ELISA were designed to detect antibodies generated against HIVusing either specific recombinant p24 gag protein as substrate antigens.96 well microtiter plates were coated at 4° C. overnight withrecombinant antigen at 2 μg/ml in PBS (phosphate buffered saline).solution using 50 μl/well on a rocking platform. Antigens consisted ofrecombinant p24 gag (Intracell). Plates were rinsed four times usingwash buffer (PBS/0.05% Tween 20) followed by addition of 200μl/well ofblocking buffer (1% Carnation milk solution in PBS/0.05% Tween-20) for 1hr at room temperature with rocking. Pre-sera and immune sera werediluted in blocking buffer at the desired range of dilutions and 100 μladded per well. Plates were incubated for 1 hr at room temperature withrocking and then washed four times with wash buffer. Secondaryantibodies conjugated with horse radish perozidase, (anti-rhesus Ig,Southern Biotechnology Associates; anti-mouse and anti-rabbit Igs,Jackson Immuno Research) diluted 1:2000 in blocking buffer, were thenadded to each sample at 100 μl/well and incubated 1 hr at roomtemperature with rocking. Plates were washed 4 times with wash bufferand then developed by addition of 100 μl/well of an o-phenylenediamine(o-PD, Calbiochem) solution at 1 mg/ml in 100 mM citrate buffer at pH4.5. Plates were read for absorbance at 450 nm both kinetically (firstten minutes of reaction) and at 10 and 30 minute endpoints (Thermo-maxmicroplate reader, Molecular Devices).

EXAMPLE 8 T Cell Proliferation Assays:

[0110] PBMCs are obtained and tested for recall responses to specificantigen as determined by proliferation within the PBMC population.Proliferation is monitored using ³H-thymridine which is added to thecell cultures for the last 18-24 hours of incubation before harvest.Cell harvesters retain isotope-containing DNA on filters ifproliferation has occurred while quiescent cells do not incorporate theisotope which is not retained on the filter in free form. For eitherrodent or primate species 4×10⁵ cells are plated in 96 well microtiterplates in a total of 200 μl of complete media (RPMI/10% fetal calfserum). Background proliferation responses are determined using PBMCsand media alone while nonspecific responses are generated by usinglectins such as phytohaemagglutin (PHA) or concanavalin A (ConA) at 1- 5μ/ml concentrations to serve as a positive control. Specific antigenconsists of either known peptide epitopes, purified protein, orinactivated virus. Antigen concentrations range from 1- 10 μM forpeptides and 1-10 μg/ml for protein. Lectin-induced proliferation peaksat 3-5 days of cell culture incubation while antigen-specific responsespeak at 5-7 days. Specific proliferation occurs when radiation countsare obtained which are at least three-fold over the media background andis often given as a ratio to background, or Stimulation Index (SI).

EXAMPLE 9

[0111] The strategies we employ are designed to induce both cytotoxic Tlymphocyte (CTL) and neutralizing antibody responses to HIV, principallydirected at the HIV gag (˜95% conserved) and env (gp160 or gp120; 70-80%conserved) gene products gp160 contains the. only known neutralizingantibody epitopes on the HIV particle while the importance of anti-envand anti-gag CTL responses are highlighted by the known association ofthe onset of these cellular immunities with clearance of primary viremiafollowing infection, which occurs prior to the appearance ofneutralizing antibodies (Koup et al., J. Virol.68: 4650 (1994)), as wellas a role for CTL in maintaining disease-free status. Although HIV isnotorious for its genetic diversity we hope to obtain greater breadth ofneutralizing antibodies both by including several representative envgenes derived from clinical isolates and gp41 (˜90% conserved; andcontains the more conserved 2F5 neutralization epitope), while thehighly conserved gag gene should generate broad cross-strain CTLresponses. Because this vaccine strategy generates both strong humoraland cellular immunity against HIV (in nonhuman primates; this approachoffers unique advantages compared to other available vaccinationstrategies for HIV.

A. HIV-1 gag Polynucleotide Vaccine Development:

[0112] Based upon our experiments for HIV env gene expression utilizinggenes comprised of optimal codons for human expression, a synthetic p55gag gene (opt gag) was designed and synthesized containing optimal codonusage throughout resulting in ˜350 silent mutations (of 1500 nt total)and cloned into V1R. A second form of opt gag vector was alsoconstructed which contained the sequence encoding the tPA signal peptideat the NH₂-terminus similar to that described above for HIV env and alsoeliminated a nuclear localization sequence motif located at thisposition in the wild type gene. This modification was designed to testwhether altering the normal intracellular trafficking patterns for gaginto the ER/golgi secretory pathway could alter the itmunogenicity ofthe gag DNA vaccine. The addition of the tPA leader peptide to gagcaused much higher levels of gag to be secreted and the secreted proteinmigrated as higher molecular weight form compared to w.t. gag. Thisindicated that posttranslation modification, probably glycosylation,occurred as a result of modification with the tPA leader peptide.

[0113] Mice that had been immunized with either one of the two optimizedp55 gag constructs (V1R-opt gag±tPA leader) or V1R-gag (wild-type) weretested for anti-gag peptide CTL responses following one injection(vaccination doses=10, 3.3, or 1 μg/mouse). High levels of anti-gag CTLwere generated by both V1-opt gag DNAs at all doses with V1R-tPA-opt gaggiving the highest specific killing (˜85% @ E/T=3 with the 1 μg dose).Comparison of cytotoxicity curves at each DNA doses demonstrated thatV1R-tPA-opt gag vaccination produced ˜100-fold stronger CTL responsesthan did V1R-gag (wild-type). Overall, immune responses for the threevaccine groups showed the same relative potencies for CTL, T help, andantibody responses (in order from greatest to lowest response):V1R-tPA-opt gag>V1R-opt gag>V1R gag (wild-type). In summary, CTL,humoral and helper T cell responses are much higher for the opt gagconstructs, especially with a tPA leader.

EXAMPLE 10 Method of Treatment

[0114] A person in need of therapeutic or prophylactic immunizationagainst infection with human immunodeficiency virus is injected with HIVDNA encoding all or part of the env, gag or pol genes or combinationsthereof. The injection may be i.p., subcutaneous, intramuscular orintradermal. The HIV DNA may be used as a primer of the immune responseor may be used as a booster of the immune response. The injection of DNAmay antedate, coincide or follow injection of the person with apharmaceutical composition comprising inactivated HIV, attenuated HIV,compositions comprising HIV-derived proteins, or combinations thereof.

EXAMPLE 11 Method of Treatment

[0115] A person in need of therapeutic treatment for infection withhuman immunodeficiency virus is treated with an anti-HIV antiviral agentor combinations thereof. The treated individual is injected with the HIVDNA pharmaceutical compositions of the instant disclosure.

1 1 1532 base pairs nucleic acid double both cDNA NO NO 1 AGATCTACCATGGGTGCTAG GGCTTCTGTG CTGTCTGGTG GTGAGCTGGA CAAGTGGGAG 60 AAGATCAGGCTGAGGCCTGG TGGCAAGAAG AAGTACAAGC TAAAGCACAT TGTGTGGGCC 120 TCCAGGGAGCTGGAGAGGTT TGCTGTGAAC CCTGGCCTGC TGGAGACCTC TGAGGGGTGC 180 AGGCAGATCCTGGGCCAGCT CCAGCCCTCC CTGCAAACAG GCTCTGAGGA GCTGAGGTCC 240 CTGTACAACACAGTGGCTAC CCTGTACTGT GTGCACCAGA AGATTGATGT GAAGGACACC 300 AAGGAGGCCCTGGAGAAGAT TGAGGAGGAG CAGAACAAGT CCAAGAAGAA GGCCCAGCAG 360 GCTGCTGCTGGCACAGGCAA CTCCAGCCAG GTGTCCCAGA ACTACCCCAT TGTGCAGAAC 420 CTCCAGGGCCAGATGGTGCA CCAGGCCATC TCCCCCCGGA CCCTGAATGC CTGGGTGAAG 480 GTGGTGGAGGAGAAGGCCTT CTCCCCTGAG GTGATCCCCA TGTTCTCTGC CCTGTCTGAG 540 GGTGCCACCCCCCAGGACCT GAACACCATG CTGAACACAG TGGGGGGCCA TCAGGCTGCC 600 ATGCAGATGCTGAAGGAGAC CATCAATGAG GAGGCTGCTG AGTGGGACAG GCTGCATCCT 660 GTGCACGCTGGCCCCATTGC CCCCGGCCAG ATGAGGGAGC CCAGGGGCTC TGACATTGCT 720 GGCACCACCTCCACCCTCCA GGAGCAGATT GGCTGGATGA CCAACAACCC CCCCATCCCT 780 GTGGGGGAAATCTACAAGAG GTGGATCATC CTGGGCCTGA ACAAGATTGT GAGGATGTAC 840 TCCCCCACCTCCATCCTGGA CATCAGGCAG GGCCCCAAGG AGCCCTTCAG GGACTATGTG 900 GACAGGTTCTACAAGACCCT GAGGGCTGAG CAGGCCTCCC AGGAGGTGAA GAACTGGATG 960 ACAGAGACCCTGCTGGTGCA GAATGCCAAC CCTGACTGCA AGACCATCCT GAAGGCCCTG 1020 GGCCCTGCTGCCACCCTGGA GGAGATGATG ACAGCCTGCC AGGGGGTGGG GGGCCCTGGT 1080 CACAAGGCCAGGGTGCTGGC TGAGGCCATG TCCCAGGTGA CCAACTCCGC CACCATCATG 1140 ATGCAGAGGGGCAACTTCAG GAACCAGAGG AAGACAGTGA AGTGCTTCAA CTGTGGCAAG 1200 GTGGGCCACATTGCCAAGAA CTGTAGGGCC CCCAGGAAGA AGGGCTGCTG GAAGTGTGGC 1260 AAGGAGGGCCACCAGATGAA GGACTGCAAT GAGAGGCAGG CCAACTTCCT GGGCAAAATC 1320 TGGCCCTCCCACAAGGGCAG GCCTGGCAAC TTCCTCCAGT CCAGGCCTGA GCCCACAGCC 1380 CCTCCCGAGGAGTCCTTCAG GTTTGGGGAG GAGAAGACCA CCCCCAGCCA GAAGCAGGAG 1440 CCCATTGACAAGGAGCTGTA CCCCCTGGCC TCCCTGAGGT CCCTGTTTGG CAACGACCCC 1500 TCCTCCCAGTAAAATAAAGC CCGGGCAGAT CT 1532

What is claimed is:
 1. A synthetic polynucleotide comprising a DNAsequence encoding a nonmammalian protein or fragment thereof, the DNAsequence comprising codons optimized for expression in a mammalian host.2. The polynucleotide of claim 1 wherein the protein is selected fromHIV proteins, HSV proteins, HAV proteins, HBV proteins, HCV proteins,HPV proteins, HSV proteins, Plasmodium proteins, Mycobacterium proteins,Borrelia proteins and rotavirus proteins.
 3. The polynucleotide of claim2 wherein the protein is an HIV protein.
 4. The polynucleotide of claim3 having the following DNA sequence: 1 AGATCTACCA TGGGTGCTAG GGCTTCTGTGCTGTCTGGTG GTGAGCTGGA (SEQ ID NO:1) 51 CAAGTGGGAG AAGATCAGGC TGAGGCCTGGTGGCAAGAAG AAGTACAAGC 101 TAAAGCACAT TGTGTGGGCC TCCAGGGAGC TGGAGAGGTTTGCTGTGAAC 151 CCTGGCCTGC TGGAGACCTC TGAGGGGTGC AGGCAGATCC TGGGCCAGCT201 CCAGCCCTCC CTGCAAACAG GCTCTGAGGA GCTGAGGTCC CTGTACAACA 251CAGTGGCTAC CCTGTACTGT GTGCACCAGA AGATTGATGT GAAGGACACC 301 AAGGAGGCCCTGGAGAAGAT TGAGGAGGAG CAGAACAAGT CCAAGAAGAA 351 GGCCCAGCAG GCTGCTGCTGGCACAGGCAA CTCCAGCCAG GTGTCCCAGA 401 ACTACCCCAT TGTGCAGAAC CTCCAGGGCCAGATGGTGCA CCAGGCCATC 451 TCCCCCCGGA CCCTGAATGC CTGGGTGAAG GTGGTGGAGGAGAAGGCCTT 501 CTCCCCTGAG GTGATCCCCA TGTTCTCTGC CCTGTCTGAG GGTGCCACCC551 CCCAGGACCT GAACACCATG CTGAACACAG TGGGGGGCCA TCAGGCTGCC 601ATGCAGATGC TGAAGGAGAC CATCAATGAG GAGGCTGCTG AGTGGGACAG 651 GCTGCATCCTGTGCACGCTG GCCCCATTGC CCCCGGCCAG ATGAGGGAGC 701 CCAGGGGCTC TGACATTGCTGGCACCACCT CCACCCTCCA GGAGCAGATT 751 GGCTGGATGA CCAACAACCC CCCCATCCCTGTGGGGGAAA TCTACAAGAG 801 GTGGATCATC CTGGGCCTGA ACAAGATTGT GAGGATGTACTCCCCCACCT 851 CCATCCTGGA CATCAGGCAG GGCCCCAAGG AGCCCTTCAG GGACTATGTG901 GACAGGTTCT ACAAGACCCT GAGGGCTGAG CAGGCCTCCC AGGAGGTGAA 951GAACTGGATG ACAGAGACCC TGCTGGTGCA GAATGCCAAC CCTGACTGCA 1001 AGACCATCCTGAAGGCCCTG GGCCCTGCTG CCACCCTGGA GGAGATGATG 1051 ACAGCCTGCC AGGGGGTGGGGGGCCCTGGT CACAAGGCCA GGGTGCTGGC 1101 TGAGGCCATG TCCCAGGTGA CCAACTCCGCCACCATCATG ATGCAGAGGG 1151 GCAACTTCAG GAACCAGAGG AAGACAGTGA AGTGCTTCAACTGTGGCAAG 1201 GTGGGCCACA TTGCCAAGAA CTGTAGGGCC CCCAGGAAGA AGGGCTGCTG1251 GAAGTGTGGC AAGGAGGGCC ACCAGATGAA GGACTGCAAT GAGAGGCAGG 1301CCAACTTCCT GGGCAAAATC TGGCCCTCCC ACAAGGGCAG GCCTGGCAAC 1351 TTCCTCCAGTCCAGGCCTGA GCCCACAGCC CCTCCCGAGG AGTCCTTCAG 1401 GTTTGGGGAG GAGAAGACCACCCCCAGCCA GAAGCAGGAG CCCATTGACA 1451 AGGAGCTGTA CCCCCTGGCC TCCCTGAGGTCCCTGTTTGG CAACGACCCC 1501 TCCTCCCAGT AAAATAAAGC CCGGGCAGAT CT.


5. The polynucleotide of claim 3 which induces anti-HIV neutralizingantibody, HIV specific T-cell immune responses, or protective immuneresponses upon introduction into vertebrate tissue, including humantissue in vivo, wherein the polynucleotide comprises a gene encoding anHIV gag, gag-protease, or env gene product.
 6. A method for inducingimmune responses in a vertebrate which comprises introducing between 1ng and 100 mg of the polynucleotide of claim 1 into the tissue of thevertebrate.
 7. The method of claim 6 which further comprisesadministration of attenuated pathogen, killed pathogen, subunitvaccines, protein vaccines and combinations thereof.
 8. An immunogeniccomposition for inducing immune responses against HIV infection whichcomprises the polynucleotide of claim 3 and a pharmaceuticallyacceptable carrier, and optionally, an adjuvant.
 9. A method forinducing anti-HIV immune responses in a primate which comprisesintroducing the polynucleotide of claim 3 into the tissue of saidprimate and concurrently administering a cytokine parenterally.
 10. Amethod of inducing an antigen presenting cell to stimulate cytotoxic andhelper T-cell proliferation an effector functions including lymphokinesecretion specific to HIV antigens which comprises exposing cells of avertebrate in vivo to the polynucleotide of claim
 3. 11. A method oftreating a patient in need of such treatment comprising administering tothe patient the polynucleotide of claim 3 in combination with ananti-HIV antiviral agent.
 12. A pharmaceutical composition comprisingthe polynucleotide of claim 1.